SEPARATION OF NEUROPEPTIDE-Y DIASTEREOMERS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND CAPILLARY ZONE ELECTROPHORESIS

Separation of analogues of neuropeptide Y (NPY) in which a single D-amino acid replaced the corresponding naturally occurring residue was performed by chromatographic techniques to ensure the quality of the synthetic peptides to be used for structural and biological studies. Of the 35 compounds, 28 were easily separated (alpha = 1.02-2.76) from native NPY by standard reversed-phase high-performance chromatography (RP-HPLC) methods using a Vydac C18 column and a gradient buffer system developed in our laboratory comprised of triethylammonium phosphate (TEAP) at pH 2.25 and acetonitrile at 40-degrees-C. The identical diastereomers could be separated on the same solid support and by using 0.1 % trifluoroacetic acid (TFA) as the mobile phase modifier, however separation factors were smaller and retention times were longer. Three of the remaining seven unresolved analogues were separated (alpha = 1.02-1.96) by changing the solid-phase support to Vydac diphenyl derivatized silica and a buffer system consisting of 0.1 % TFA and acetonitrile. Of the four remaining unresolved analogues, only two could be separated by capillary zone electrophoresis (CZE) in 0.1 M sodium phosphate at pH 2.5, but all four were finally resolved by changing the electrophoretic buffer to 0.1 M TEAP buffer at pH 2.5. Migration times of the diastereomers differed by 0.2-2.0 min from that of the natural NPY. In addition to confirming the uniqueness of each isomer, this investigation demonstrated the expansive utility and high efficiency of the TEAP buffer system for both RP-HPLC and CZE as well as the difference in selectivity produced by the TEAP and TFA buffers in RP-HPLC. The conditions described here have broad applications for the analysis and preparative separation of synthetic and native peptides.